The present invention relates to the field of automated analyzers for nucleic acid diagnostics, in particular to temperature control devices. It is well known in the field of molecular biology that a reaction is influenced by the temperature at which the reaction is performed. If the temperature of the reaction varies, the results could be inconsistent with previous assays or with results of the calibration reactions. Precise temperature control to provide heating and cooling cycles is useful in many processes and particularly useful in gene amplification and detection processes.
This invention more fully describes an embodiment of the carrier which incorporates a standard microscope slide as part of the carrier as described in U.S. Pat. No. 5,188,963 and copending U.S. patent application Ser. No. 836,348; and PCT US/90/06768. This invention describes a carrier assembly of multiple carriers which uses a slide as the main portion of each carrier bottom and frames the edges which fit around the slide in keeping with the original carrier format. This invention describes the automated apparatus's temperature control system and its integration with the carrier for more precise temperature regulation.
The in situ amplification process (described in U.S. Pat. No. 5,188,963, and copending U.S. patent applications, Ser. No. 227,348; Ser. No. 836,348; and PCT/90/06768) uses enzymes such as polymerase or ligase, separately or in combination, to repeatedly generate more copies of a target nucleic acid sequence by primer extensions to incorporate new nucleotides or by ligations of adjacent complementary oligonucleotides, wherein each template generates more copies and the copies may themselves become templates. By melting complementary strands of nucleic acids, the original strand and each new strand synthesized are potential templates for repeated primer annealing or ligation reactions to make and expand the number of specific, amplified products. A thermostable polymerase with reverse transcriptase activity and a thermostable ligase are now both available and increase the choice of enzymes and combination of reactions for in situ applications. As stated in copending U.S. patent application Ser. No. 227,348, if RNA in the specimen is the target to be amplified, the specimen is treated with reverse transcriptase to make a nucleic acid complement of the RNA just prior to amplification. Using a thermostable reverse transcriptase polymerase such as rTth (Perkin Elmer, Norwalk, Conn.), it may not be necessary to add another polymerase for rounds of primer extension amplification. The amplification can either be primer extensions in one direction for linear amplification, or in opposing directions, for geometric amplification. The label can either be incorporated as labeled nucleotides or labeled primers for one-step detection or labeled probes may be added in a step following amplification whereby the probes hybridize to the amplified products for detection.
Nucleic acid amplification had been limited to solution reactions wherein the nucleic acid is released from cells or tissue. In U.S. Pat. No. 5,188,963 and copending U.S. patent application Ser. Nos. 227,348 and 836,348, a process to amplify nucleic acid targets within cells was described and a method for embedding the cellular specimens in a matrix was described to immobilize and stabilize the cells during amplification and detection. A number of examples for using in situ amplification are given in U.S. Pat. No. 5,188,963. A photomicrograph of cells which had amplified and labeled DNA was included in Ser. No. 836,348 to show that the amplified fragments are retained in individual cells and such cells can be enumerated under microscopic observation.
The process requires at least one denaturing or high temperature stage, and one primer annealing or low temperature stage in each cycle. To achieve the desired results, the embedded cell specimens are heated to nucleic acid denaturation temperature and temperature control commences before reagent addition. Since the specificity of nucleic acid hybridization is influenced by temperature, uniform and accurate temperature for all specimens is maintained throughout the reaction. The time required for the specimen to be brought to the reaction temperatures can be a large percentage of the time allowed for the biochemical processes to be performed; therefore, means to cycle temperature rapidly and reliably are desirable.
There are various techniques and devices for adjusting temperature of reagents and specimens thereafter controlling the reaction temperature. For example, it is known to use individual reaction heating coils around individual reaction vessels. While a circulating air or water bath can control temperature of a large number of reactions simultaneously, the rate at which heat transfers from such a bath to a reaction vessel is substantially proportional to the difference between the temperature of the vessel and the temperature of the bath, to the heat capacity of the fluid, and to the efficiency of the contact therebetween. (See, for example, U.S. Pat. No. 5,038,852 where circulating fluid reservoirs or Peltier heat pumps are described for heating and cooling a reaction mix.) The specific heat of air is so small that it becomes very difficult to control the temperature of reaction vessels accurately in circulating air. While water has a superior specific heat compared to air, it must be moved rapidly about the reaction vessels to maintain narrow temperature tolerances and, unfavorably, the water supports microbial growth. In addition to fluid baths, it is also commonly known to install reaction vessels in thermal contact with a temperature controlled body or mass having good thermal conductivity and a specific heat as high as practical. For example, a plurality of reaction vessels may be located within an aluminum or copper body.
The aforementioned in situ amplification for cellular analyses, which requires precise temperature regulation, creates a need for an improved apparatus which adjusts and controls the temperature of the cellular specimens An apparatus designed for rapid temperature cycling necessitates reducing thermal loads to increase the rate at which heat transfers occur. The carriers used in this invention are thin, flat reaction vessels whose bottom piece transfers and spreads the heat quickly to the ultra-thin specimen within. Using the word "thin" herein for carrier means that the carrier bottom that conducts heat to the specimen is preferably not thicker than 1 millimeter. Using the word "ultra-thin" herein for specimen means that a rehydrated matrix and specimen is preferably not thicker than 0.5 millimeter. Because the specimen is ultra-thin and represents a significantly greater surface area to volume ratio than what would be found in a conical tube, the specimen temperature more closely matches the temperature of the bottom piece. For e.g., the surface area-to-volume ratio of 100 microliters in a conical tube is 132:1; whereas, the surface area-to-volume ratio in a flat carrier (with a 2 cm.times.2 cm matrix and specimen holding area) holding an equivalent 100 microliter sample is 830:1, or more than six times greater. For example, a conical microfuge tube filled to a depth of 1 centimeter at a maximum width of 0.62 centimeters has a surface area of 1.32 cm.sup.2 and a volume of 0.1 cubic centimeter (100 microliters). A carrier with a sample 2 cm.times.2 cm.times.0.025 cm also has volume of 0.1 cubic centimeter, but has a surface area of 8.3 cm.sup.2.
When glass slides are inserted in a carrier assembly as separate carrier bottoms, each glass slide becomes part of the heat flow transfer to and from a specimen. A specimen in the thin, aforementioned configuration has greater surface contact with the slide (carrier bottom), thereby reflecting quicker temperature changes with respect to the flat carrier bottom, than a specimen-containing solution with respect to the aforementioned conical tube. Using glass in the bottom carrier piece, or a material with comparable heat conductivity characteristics, also improves the heat transfer capability of the carrier format over standard microfuge tubes made of polypropylene. A flat configuration of the matrix and specimen holding area on a carrier enables convenient microscopic analysis of molecular targets within the individual cells immobilized throughout the specimen.
Discrimination between binding specificity of different nucleic acid primers and probes to target molecules is affected by temperature. Minor sequence variations in nucleic acid base composition may be detected within individual cells either by labeling newly-incorporated nucleotides from specific oligonucleotides and/or amplifying the target sequence and then hybridizing a labeled probe to the amplification products. These sequence variations may be used in DNA-based diagnostics to identify infectious disease, genetic disease, cancer or identity-testing. Precise temperature control is required to use genetic sequence information most fully and produce exquisitely accurate results.
The object of the invention is to provide an apparatus and method of accurately controlling the temperature of simultaneous biochemical reactions, bringing all the individual reactions to a desired temperature, holding the reactions to the specified temperature for a period of time, cooling the reactions to a desired temperature, and holding the reactions at the specified temperature for a period of time. Further objects, features and advantages of the invention will become apparent from a consideration of the following description, taken in conjunction with the accompanying drawing figures.